PB2061: A NEW DIAGNOSTIC TOOL BASED ON MOLECULAR APPROACHES APPLICABLE TO ROUTINE CLINICAL USE FOR FANCONI ANEMIA PATIENTS ORIGINATING FROM NORTH AFRICA
Authors: Olfa Kraiem, Abir Ben Haj Ali
Journal: HemaSphere (2023)

Topic: 12. Bone marrow failure syndromes incl. PNH - Clinical Background: Fanconi anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents and cancer predisposition; in 2020, the Tunisian Fanconi Anemia Registry includes 150 patients. FA-A complementation group is the most prevalent (94%). A common haplotype shared by FA patients mostly originating from the South of Tunisia was associated with the deletion of exon 15 in FANCA. Aims: The present study aims to investigate the FANCA mutational spectrum and to develop a strategy based on molecular approaches applicable to routine clinical use for FA patients originating from North Africa. Methods: This study was conducted on 22 FA patients. Genomic DNA was extracted using the standard salting-out method or the QIAamp DNA mini kit (Qiagen). Clinical data were obtained from the Tunisian fanconi anemia registry TFAR. Patients were initially screened using PCR to check the homozygous deletion of the exon 15 in the FANCA gene. The 43 coding exons of FANCA gene were amplified using PCR. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large deletions or duplications within the FANCA gene. Targeted gene sequencing (TGS) has been conducted for four patients. Results: In this study, we report on the clinical, cytogenetic, and molecular investigation of 15 families including 22 FA patients. Our cohort is composed of 6 girls and 16 boys (sex ratio = 2.66) aged from 1 to 28 years. Inbreeding examination showed that 19 patients (86.36%) were born from consanguineous unions. Eight families are multiplex. Geographical distribution showed 14 patients originating from Southern Tunisia, six from Central Tunisia and two from Northern Tunisia. Most of patients presented typical clinical features of FA including: skin pigmentation abnormalities (86%), microcephaly (52%), triangular face (31%), and skeletal malformations (34%). Other clinical abnormalities, such as ear abnormalities and hearing problems were presented in two patients. Chromosomal breakage analysis was successful in all patients, the cytogenetic results revealed a high frequency of chromosomal breakages. The molecular approach allowed us to identify seven different pathogenic variants at homozygous and heterozygous states. These variants include large deletions of one or two exons (deletion of exon 15, deletion of exons 4 and 5) respectively, one nonsense mutation in exon 28, a small deletion in exon 38, one likely pathogenic missense variant in exon 14 that was already associated to FA, a previously reported intronic in intron 24, and a novel missense variant of uncertain significance in exon 14. The last intronic mutation was inherited at a homozygous state in all FA patients originating from Kairouan (Central Tunisia). Furthermore, all these patients shared the same haplotype. All patients from Sidi Bouzid and Gafsa were homozygous for the founder mutation, deletion of exon 15, except for one patient originating from Gafsa who was homozygous for the novel missense variant. Deletion of exons 4 and 5 has been identified in five patients originating from Sfax. Four of them belong to two related families and were homozygous for this large deletion. The other patient was heterozygous and the second mutation is still unknown. Our findings showed that deletion of exon 15, the large deletion of exons 4 and 5, and the intronic mutationc are founder mutations in Sidi Bouzid/Gafsa, Sfax, and Kairouan, respectively (figure 1). Summary/Conclusion: The assignment of NA patients to their genetic FA subtypes and the identification of their respective mutations provide valuable data for a better knowledge of the genetic basis of FA in NA and for a more appropriate management of patients at both and therapeutic levelsKeywords: Fanconi anemia, FANCA, Molecular cytogenetics